The Preliminary Data show that the calcium-sensing receptor (CaR) suppresses parathyroid hormone-related protein (PTHrP) production by mammary epithelial cells (MEC) during lactation, and increases calcium (Ca) transport into milk. We will first use CaR-null mouse models to confirm these data. We will use graded dietary Ca restriction and calcimimetics to examine the CaR-mediated effects of systemic Ca on PTHrP production in the mammary glands (MG) of wild type mice, CaR mice, and CaR-/- mice made viable by the genetically engineered absence of parathyroid glands (CaR-/-/Gcm2-/-). We will also use this approach to study the regulation of MG Ca transport into milk by the CaR. We will examine the effect of extracellular Ca and calcimimetics on PTHrP production by MEC from these mice in culture, and characterize the signaling pathways, downstream from the CaR, that regulate PTHrP. Next, we will use three-dimensional cultures of primary MEC from the CaR-null mice, to examine the regulation of transepithelial Ca transport by the CaR, using a 45Ca tracer. We will also describe the signaling pathways that couple the CaR to Ca transport in MEC. Finally, in order to study the CaR's role in the MG during lactation on a backdrop of normal Ca metabolism, we will use the cre-lox system to delete the CaR specifically in the lactating MG. Using this model, we will study the role of the CaR in regulating PTHrP production, Ca transport, and milk production in the MG. We will also study systemic Ca and bone metabolism in these mice during lactation, as these are targets of mammary-derived PTHrP action. My career aim is to develop a research program at the intersection of Ca metabolism and MG biology that will lead to independence. In the short term, I would like to acquire skills in cell signaling and Ca imaging to complement my expertise in mammary gland biology. This will be accomplished through formal course work and by interactions with the co-sponsor and collaborators. Yale is an ideal training environment, with an abundance of physical and intellectual resources.